Serum protein electrophoresis in European mink (Mustela lutreola): reference intervals and comparison of agarose gel electrophoresis and capillary zone electrophoresis.

BACKGROUND: Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species. MATERIAL AND METHODS: Serum samples were obtained from European minks enrolled in the breeding program (n = 55). Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) were used to separate and identify protein fractions. Albumin, α1, α2, ß, and γ-globulins fractions were identified in all mink sera by both electrophoresis methods. Reference intervals (90% CI) were determined following the 2008 guidelines of the Clinical Laboratory Standard Institute. The methods were compared using Passing-Bablok regression, Bland-Altman analysis, and Lin's concordance correlation. RESULTS: A significant bias was found between methods for α1, α2, and γ-globulin. Lin's concordance correlation was considered unacceptable for α1, α2, and ß-globulins. Differences for gender between methods were found for albumin and α2-globuins, which were higher for males than females. γ-globulins were higher for adults than young minks using both methods; however, α1 and α2-globulins were lower. CONCLUSION: Both methods are adequate for identifying serum protein disorders, but the AGE and CZE methods are not equivalent. Therefore, reference intervals for each technique are required.

A Pilot Study Investigating Plasma Protein Electrophoresis in One Anuran and Six Urodelan Species.

As threats to amphibian health increase, there is a growing need for diagnostic tools to assess and monitor their health status. Plasma protein electrophoresis has proven to be useful in other nonmammalian species. It enables quantification of protein fractions in plasma that may be altered in various disease processes, and is therefore useful in narrowing down differential diagnoses and detecting inflammation, in combination with other modalities such as biochemical and hematologic testing. The amphibian electrophoretogram must be defined before baseline reference intervals are obtained across species. Agarose gel electrophoresis was performed on plasma samples collected from presumed clinically normal individuals of one anuran and six urodelans: Osteopilus septentrionalis (n=2), Gyrinophilus porphyriticus (n=1), Notophthalmus viridescens (n=1), Eurycea guttolineata (n=2), Amphiuma tridactylum (n=2), Cryptobranchus alleganiensis (n=5), and Siren lacertina (n=6). The electrophoretograms varied in number of fractions between each species; however, the number of fractions was consistent within a species. An albumin migrating fraction was consistently observed in all species. A prealbumin migrating fraction was identified in species that primarily use organs other than skin for respiration. This study provides preliminary examples of a normal plasma protein electrophoretogram for seven amphibian species. Further studies quantifying reference intervals and identification of protein fractions will help establish protein electrophoresis as a useful tool in amphibian health investigations.

Comparison of agarose gel and capillary zone electrophoresis methods with preliminary reference interval generation using capillary zone electrophoresis in bald eagles (Haliaeetus leucocephalus).

BACKGROUND: Electrophoresis can be used to aid in the diagnosis of different diseases in avian species. Capillary zone electrophoresis (CZE) is an automated method that is proposed to be superior to the dye methods used in agarose gel electrophoresis (AGE). However, reference intervals (RIs) for CZE in avian species and comparison studies between electrophoretic methods are lacking. OBJECTIVES: The goals of the current study were to compare AGE and CZE methods and determine reference intervals for CZE using plasma from bald eagles (Haliaeetus leucocephalus). METHODS: Heparinized plasma samples from 44 bald eagles (mean age 18.7 years) under managed care were examined by AGE and CZE. Method comparison analyses were completed, as well as the generation of preliminary RIs using the CZE method and ASVCP guidelines. RESULTS: Passing-Bablok regression and Bland-Altman plots demonstrate that these methods are not equivalent. All fractions were significantly correlated between the methods except for alpha 1 globulin. Inter-assay and intra-assay CVs for CZE were lower or comparable to AGE and ranged from 2.4% to 15.4%, and 0.8% to 8.3%, respectively. CZE resolved more fractions than AGE with two fractions observed in the beta and gamma region vs one for AGE in each region. CONCLUSIONS: CZE provided improved resolution and reproducibility for the quantitation of protein fractions in the bald eagle. Although most fraction results correlated with AGE, these methods were judged as not equivalent, necessitating method-specific Rls. Reference intervals generated from a limited number of mostly aged individuals under managed care should be considered preliminary; additional studies will aid in the production of more robust intervals.

Agarose gel electrophoresis pattern of serum creatine kinase and lactate dehydrogenase isoenzymes in zoo-managed Asian elephants (Elephas maximus).

Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzymes were evaluated in nine zoo-managed Asian elephants (Elephas maximus) using a commercial agarose gel electrophoresis (AGE) kit. CK was separated into two major fractions, CK-BB and CK-MM, along with a small fraction of macroenzyme-CK type 2 (mCK2); CK-MM was the largest fraction. LDH was separated into five fractions (LDH1-5); LDH3 was the largest fraction. Age was negatively and positively correlated with the percentages of CK-BB and CK-MM, respectively, and negatively correlated with CK-BB and mCK2 activities. These results indicate that an AGE kit can be used to evaluate CK and LDH isoenzymes. Routine isoenzyme testing may enable early detection of disease and physiological changes.

ASSESSMENT OF CAPILLARY ZONE ELECTROPHORESIS AND SERUM AMYLOID A QUANTITATION IN CLINICALLY NORMAL AND ABNORMAL SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM) AND SOUTHERN BLACK RHINOCEROS (DICEROS BICORNIS MINOR).

Capillary zone electrophoresis (CZE) and an immunoassay for serum amyloid A (SAA) were used to examine serum samples from clinically normal and abnormal southern white rhinoceros (Ceratotherium simum simum) and southern black rhinoceros (Diceros bicornis minor) under managed care. CZE resolved seven fractions as well as subfractions for α1 globulins. Reference intervals were calculated for white rhinoceros (n = 33) and found to have some differences over previously reported intervals generated using agarose gel electrophoresis (AGE) methods in sera from free-ranging animals. In addition, the coefficient of variation related to fraction quantitation was found to be overlapping or superior to that reported for AGE. No significant differences were observed in CZE measurands and total protein between clinically normal and abnormal rhinoceros. In contrast to CZE, significant differences in SAA levels (P < 0.001) were observed in samples from the white rhinoceros between clinically normal and abnormal animals. In addition, in limited sample sets with repeated measures, SAA provided prognostic value. Future studies should generate more robust reference intervals and delineate the application of both SAA quantitation and CZE in routine health assessments and in prognostication.

Protein electrophoresis of non-traditional species: A review.

EPH has been demonstrated to be a useful tool in companion animals while providing an opportunity to characterize globulinemias, including paraproteinemia. In EPH of non-traditional species, these same applications are important, but the primary use is to gauge the acute-phase and humoral immune responses. This includes the valid quantitation of albumin as well as the examination of fractions reflective of increases in acute-phase reactants and immunoglobulins. Agarose gel EPH and, more recently, capillary zone EPH have been applied to samples from these species. Performing these analyses provides special challenges in the placement of fraction delimits, generation of RIs, and interpretation of results. Recommended as part of routine bloodwork, EPH can also provide key results that are helpful in clinical and field-based health assessments as well as in prognostication.

Method comparison for serum protein electrophoretic M-protein quantification: Agarose gel electrophoresis and capillary zone electrophoresis in canine and feline sera.

BACKGROUND: Densitometric quantification of myeloma paraproteins (M-proteins) is used to monitor secretory myeloma related disorders in humans and dogs. The previous work in dogs used agarose gel electrophoresis (AGE) but did not establish if other methods of serum protein electrophoresis, such as capillary zone electrophoresis (CZE), were comparable. OBJECTIVES: We aimed to determine if the densitometric quantification of M-proteins using CZE would yield results comparable to AGE methods. METHODS: Fifty-one serum samples from 22 dogs and 18 cats with confirmed monoclonal gammopathies and previously performed AGE were evaluated using CZE on a Sebia Minicap system. Samples were run in duplicate, and their M-proteins were densitometrically measured using the corrected perpendicular drop method previously described. Human-based quality control samples were used to determine the inter-run coefficient of variation (CV). Patient samples were used to calculate the intra-run CV. Method comparison was performed using simple linear regression, Passing-Bablok regression, and Bland-Altman analyses, and Medx evaluations. RESULTS: Inter-run and intra-run CVs for CZE were 3.71%-7.65% and 2.89%-4.74%, respectively. Simple linear regression demonstrated an excellent correlation (r > 0.98). Passing-Bablok regression was compatible with the presence of proportional bias in the entire population, and Bland-Altman plots revealed a proportional bias in the feline cases. The Medx evaluation suggested that the two methods did not perform similarly in clinical samples with poor performance at a decision limit of 0.5 gm/dL. CONCLUSIONS: Capillary zone electrophoresis is an acceptable method for M-protein densitometric quantification in canine and feline sera but cannot be used interchangeably with AGE-based evaluations.

THE INFECTION AND SPECIES IDENTIFICATION OF CANINE BABESIA SPP. IN PARTS OF SHAANXI PROVINCE.

Canine babesiosis is a serious disease among tick-borne haemoprotozoan diseases that threaten dog health. To find out the prevalence of canine babesiosis and its main pathogenic species in Shaanxi Province, the study was centered on the infection of babesiosis in dogs in different regions of the Province. First, a total of 367 blood samples were collected in Shaanxi Province, and 53 Babesia nucleic-acid-positive samples were found by polymerase chain reaction (PCR) identification, with a positive rate of 14.44%, and Babesia gibsoni was found by sequencing analysis. Further analysis showed that the prevalence of canine babesiosis was significantly different in 5 regions. There was no significant difference in infection rates between age groups, with the lowest prevalence in young dogs (10.81%) and the highest in adult dogs (17.29%). The infection rate in male dogs was higher than in female dogs. The morbidity of canine Babesia spp. was significantly different between different seasons, with the highest infection rate in autumn (27.78%) and the lowest in winter (6.10%). In conclusion, the epidemicity of canine Babesia spp. in dogs was mainly affected by region and season, and B. gibsoni was the most common canine Babesia spp. within Shaanxi Province in our study. These results provide basic data for the prevention and control of canine babesiosis in this region.

PARTIAL HEMATOLOGY, BIOCHEMISTRY, AND PROTEIN ELECTROPHORESIS REFERENCE INTERVALS FOR CAPTIVE SPOTTED TURTLES (CLEMMYS GUTTATA).

Spotted turtles (Clemmys guttata) are an endangered species and are commonly encountered in the pet trade and in many zoological collections across the United States, yet peer-reviewed published reference intervals (RI) for common clinicopathologic tests are unavailable for this species. The objectives of this study were to calculate partial RI for routine hematology, biochemistry, and electrophoretic analyses, as well as to perform an initial comparison of capillary zone electrophoresis (CZE) and agarose gel electrophoresis (AGE) in this species. A single blood sample was obtained from a single collection of 32 apparently healthy captive spotted turtles weighing at least 100 g and was submitted for standard hematologic and biochemistry analyses, as well as electrophoresis via CZE and AGE methods. Partial RI were calculated for corresponding analytes for each type of testing. While CZE and AGE protein fractions were found to have good correlation, some significant differences were observed, reinforcing that RI should be reported with the specific method used for their determination. The spotted turtle electrophoretograms were distinctly different from those previously reported from turtles in the same taxonomic family, including differences in the number and relative prominence of protein fractions.

Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 1 - Current performance.

BACKGROUND: Routine electrophoresis [agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE)] and species-specific immunofixation (IF) can be used alone or in combination to detect immunoglobulin paraprotein (M-protein) and diagnose secretory myeloma-related disorders (sMRD). OBJECTIVE: We aimed to evaluate the performance of AGE, CZE, CZE plus IF (CZE-IF), and AGE plus IF (AGE-IF) for detecting canine serum M-proteins. METHODS: One hundred canine cases that had AGE, CZE, and routine IF performed on serum, and where B-cell lineage neoplasia (such as B-cell lymphoma and plasma cell tumors) had been diagnosed or excluded, were evaluated. Routine IF protocols targeted IgG-FC, IgA, and IgM heavy chains and light chains. IgG4 IF and free light chain IF were also performed. B-cell lineage neoplasms with an M-protein detected, using any available method, were classified as sMRD. Datasets from AGE, CZE, IF, CZE-IF, and AGE-IF (electrophoretograms, gel images, and fraction concentrations) were composed and reviewed. The sensitivity, specificity, and Youden's index for M-protein detection were determined for each dataset. RESULTS: The combination of AGE-IF or CZE-IF was more sensitive (82.9%) than CZE alone (72.0%) or AGE alone (64.6%) and more specific (66.1%, 48.3%, 51.7%, respectively). Immunofixation could be used alone to detect M-proteins (sensitivity 82.9%, specificity 61.9%), but there were technical challenges that complicated the performance and evaluation of the test. Myeloma with free light chains only was found in 5/41 cases of sMRD. CONCLUSIONS: Adding routine IF to routine electrophoresis increases the ability to accurately identify M-proteins; however, there is still room for further diagnostic performance improvements.

MOLECULAR INVESTIGATION AND PHYLOGENETIC ANALYSIS OF ANAPLASMOSIS IN DOGS.

Anaplasmosis is a widespread vector-borne disease affecting dogs, and Anaplasma platys is the major etiological agent of the disease. The study examines anaplasmosis molecular prevalence, related risk factors, and alteration of hematological variables in Anaplasma-affected dogs. A total of 150 blood samples were collected from dogs in the district of Lahore, Pakistan. The samples were screened with PCR targeting the 16S rRNA gene of Anaplasma. Sequencing of samples that were found positive after performing PCR was conducted. A questionnaire was developed to collect epidemiological data on subject dogs, and the information was analyzed with a logistic regression model using SPSS. The current study revealed an 11.34% (17/150) prevalence of anaplasmosis in dogs based on PCR detection. Tick infestation, previous tick history, house hygiene, and tick control status were major risk factors linked with disease occurrence. Red blood cell count, packed cell volume, hemoglobin, and platelet count were decreased significantly (P < 0.05) in Anaplasma-infected dogs. Phylogenetically, the 2 isolates of the current study clustered together, and that cluster was very similar to A. platys isolates from India, Malaysia, and Thailand.

COMPARISON OF AGAROSE GEL AND CAPILLARY ZONE ELECTROPHORESIS METHODS USING PLASMA FROM GREEN TURTLES (CHELONIA MYDAS).

Agarose gel electrophoresis (AGE) has been widely implemented throughout veterinary medicine and for analysis of plasma proteins of avian and reptile species. Capillary zone electrophoresis (CZE) is becoming a standard method in human clinical pathology laboratories but has not widely been used for the analysis of animal samples. The objective of the present study was to compare protein fractions derived from AGE and CZE methods using plasma from the green turtle (Chelonia mydas). Plasma samples were analyzed by AGE and CZE per manufacturer guidelines. The methods were assessed by CV analysis, Spearman's correlation, Passing-Bablok regression, and Bland Altman plots. CZE consistently resolved more fractions than AGE with three fractions observed in the prealbumin migrating region versus one for AGE and two fractions in the γ globulin region versus one for AGE. Compared with AGE, CZE showed a lower CV in intra-assay tests (1.0-4.9% vs 2.0-28.3%) and a lower or overlapping CV in interassay tests (1.0-10.6 vs 2.3-22.0). The prealbumin, α2 globulin, and ß globulin fractions correlated the least between the methods (for all three fractions: rs ≤ 0.28, P > 0.21). Moderate, significant correlations between AGE and CZE methods were observed for albumin (rs = 0.78, P < 0.0001) and γ globulins (rs = 0.78, P < 0.0001). CZE has a higher precision and ease of use over AGE and offers the opportunity to resolve additional protein fractions. This will necessitate the development of new conventions in placement of fraction delimits, definition of species-specific reference intervals, and evaluation of clinical utility in abnormal turtles.

Alterations in serum protein electrophoresis profiles during the acute phase response in dogs with acute pancreatitis.

The quantification of serum proteins is a useful tool for diagnosing and monitoring various diseases that involve changes in the concentrations of these proteins. As canine acute pancreatitis (AP) accompanies the systemic inflammatory response syndrome, serum proteins such as C-reactive protein (CRP) have been used as inflammatory markers for dogs with AP. The goal of this study was to investigate the overall profiles of serum proteins by serum protein electrophoresis (SPE) and to determine the concentration of acute phase proteins (APPs) in dogs with AP in order to better understand serum protein profiles as diagnostic markers in these dogs. Decreased levels of albumin and increased levels of alpha-2 globulin were observed in dogs with AP by SPE. Among APPs, elevated concentrations of CRP, serum amyloid A (SAA), and haptoglobin were detected. The concentration of SAA was positively correlated with that of CRP, which suggests that SAA could be a sensitive marker of inflammation in dogs with AP, similar to CRP.

La quantification des protéines sériques est un outil utile pour diagnostiquer et suivre différentes pathologies qui impliquent des changements dans les concentrations de ces protéines. Comme la pancréatite aiguë (AP) accompagne le syndrome de réponse inflammatoire systémique, les protéines sériques telles que la protéine C-réactive (CRP) ont été utilisées comme marqueurs d'inflammation chez les chiens avec AP. L'objectif de la présente étude était d'examiner les profils globaux des protéines sériques par électrophorèse des protéines sériques (SPE) et de déterminer les concentrations des protéines de phase aiguë (APPs) chez les chiens avec AP afin de mieux comprendre les profils de protéines sériques comme marqueurs diagnostiques chez ces chiens. Des niveaux diminués d'albumine et des niveaux augmentés de globuline alpha-2 furent observés par SPE chez des chiens avec AP. Parmi les APPs, des concentrations élevées de CRP, d'amyloïde sérique A (SAA), et d'haptoglobine furent détectées. La concentration de SAA était corrélée positivement avec celle de CRP, ce qui suggère que SAA pourrait être un marqueur sensible d'inflammation chez les chiens avec AP, de manière similaire à la CRP.(Traduit par Docteur Serge Messier).

Bias between two methods of albumin measurement in the white rhinoceros, Ceratotherium simum.

BACKGROUND: The bromocresol green (BCG) method has been reported to overestimate serum albumin concentration in several species due to non-specific binding to globulins. As the white rhinoceros has high concentrations of serum globulins, significant differences in albumin measured by the BCG method, and the field method of agarose gel serum protein electrophoresis (SPE) are expected. OBJECTIVES: We aimed to compare the BCG and SPE methods for albumin determination in the serum of white rhinoceroses. METHODS: SPE and BCG albumin were measured in 82 white rhinoceros serum samples. Results were compared using Bland-Altman difference plots and Passing-Bablok regression analysis. RESULTS: BCG albumin showed a significant mean constant positive bias of 7 g/L, or 36%, which was more than the total allowable error of 15% and was clinically significant. Methods were not comparable within the inherent imprecision of each method. CONCLUSIONS: The BCG method overestimated albumin concentrations in this species compared with agarose gel SPE, and method-specific reference intervals should be used.

Electrophoretic patterns of proteinuria in feline spontaneous chronic kidney disease.

OBJECTIVES: The purpose of this study was to describe the electrophoretic patterns of proteinuria in cats at risk of and cats with chronic kidney disease (CKD), and to investigate whether the presence of high-molecular-weight (HMW) and low-molecular-weight (LMW) proteins were associated with CKD, proteinuria and/or disease progression. METHODS: Healthy cats at risk of developing renal disease (n = 17) and cats affected with CKD at different stages (n = 22) were prospectively enrolled and sampled over time. Seventy urine samples were included and assayed with a commercially available sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) method. Each sample (gel lane) was inspected to identify albumin, HMW and LMW proteins, and an electrophoretic pattern (albuminuria, glomerular, tubular, mixed or negative) was assigned accordingly. Fisher's exact test was used to assess the distribution of HMW and LMW proteins in cats grouped according to International Renal Interest Society stage and to the magnitude of proteinuria, and to assess if HMW and LMW proteins at the time of inclusion were associated with the development and progression of CKD. RESULTS: In samples of cats at risk, the most common pattern was glomerular (84.6%); glomerular pattern was also common in cats with CKD (54.2%), although mixed proteinuria and tubular proteinuria were also present (29.5% and 11.4%, respectively). The presence of LMW proteins was associated with CKD (P <0.0001) and to a urine protein:creatinine ratio >0.2 (P = 0.025). Both HMW and LMW proteins were not associated with progression of CKD within 6 months (n = 14). CONCLUSIONS AND RELEVANCE: Our results showed that HMW proteinuria is common in healthy cats at risk of developing CKD, although the pathological significance needs to be confirmed. The detection of LMW proteins in urine of cats suspected to be affected by CKD, especially in non-azotaemic, non-proteinuric or borderline proteinuric cats, suggests the presence of kidney damage.

Determination of plasma bone-specific alkaline phosphatase isoenzyme activity in Holstein calves using a commercial agarose gel electrophoresis kit.

We measured the bone-specific alkaline phosphatase (ALP) isoenzyme activity in 67 plasma samples from 14 newborn Holstein calves using both a conventional method (featuring heat inactivation) and a commercial agarose gel electrophoresis (AGE) kit; the relevant isoenzymes were termed bone-specific ALP (BAP) and ALP isoenzyme 3 (ALP3). We explored whether the AGE kit afforded reliable data when used to analyze samples from Holstein calves. The blood was collected from the jugular vein of each calf immediately prior to the first colostrum feeding (pre-feeding), 20 and 40 h after pre-feeding, and on days 4 and 7; whereas three samples (from three calves) were not obtained. The total plasma ALP activity varied widely, exceeding the ranges of reference values. On electrophoresis, 52 of 67 plasma samples (77.6 %) clearly contained both ALP isoenzyme 2 and ALP3, as did control human serum. The total ALP activity of the 52 samples ranged from 166-1989 U/L (median: 1013 U/L), whereas the values for the other 15 samples (22.4%) exhibiting abnormal isoenzyme fractionation ranged from 1014-5118 U/L (median: 1780 U/L). In the 52 plasma samples exhibiting clearly separated isoenzymes, ALP3 and BAP activities were strongly positively correlated as revealed by Deming regression (y = 0.93x + 22.6, p⟨0.0001) and Bland-Altman analysis (ALP3/BAP activities limit of agreement: -5.1%). Thus, the AGE kit yields useful information on newborn calves, and can replace the conventional method when the total plasma ALP activity is less than approximately 1000 U/L.

Serum protein concentrations and protein fractions in clinically healthy Italian Heavy Draft Horses using agarose gel electrophoresis.

BACKGROUND: Serum protein electrophoresis (SPE) reference intervals (RIs) have been evaluated in different horses, but no specific values are shown for equine breeds as previously described in other species (dogs, cats), and no studies have been performed on SPE in draft horses. OBJECTIVES: This study aimed to determine RIs for SPE in heavy draft horses (Italian Heavy Draft Horse-IHDH) living in central Italy. A comparison between different physiologic states (pregnancy and no pregnancy) and ages (foals and adults) was executed. MATERIALS AND METHODS: Blood samples were collected from 215 apparently healthy horses (mares, stallions, and foals). SPE (total proteins, albumin, α1-, α2-, ß1-, ß2-, and γ-fractions, A/G) was evaluated in the Veterinary Teaching Hospital of the University of Perugia. RIs were determined using well-described, modern analytical and statistical methods. The normality of distributions was assessed using the Anderson-Darling test. Differences between subgroups were evaluated using the Mann-Whitney U test. A P < .05 was considered statistically significant for all analyses. RESULTS: Our results showed that IHDHs had increases in TPs and the α2-, ß1-, ß2-, and γ-fractions, and decreases in albumin, α1-globulins, and A/G ratios compared with the data reported in other horses. We also found that foals had significantly higher α1-globulins and significantly lower albumin concentrations, and A/G ratios compared with those of the adult horses. CONCLUSIONS: In the present study, SPE RIs using agarose gels have been determined for the first time in a large number of draft horses (represented by IHDH). The obtained results provide a basis for the further investigation of equine breeds with natural breeding, and the impact of age and physiologic states on SPE.

Molecular detection of Anaplasma phagocytophilum in wild carnivores in north-eastern Poland.

BACKGROUND: Anaplasma phagocytophilum is an obligate parasitic intracellular bacterium. It is the causative agent of granulocytic anaplasmosis, with effects on human and animal health. In Europe, the pathogen is mainly transmitted among a wide range of vertebrate hosts by blood-sucking arthropods. The aim of this study was to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dogs (Nyctereutes procyonoides), badgers (Meles meles), foxes (Vulpes vulpes), martens (Martes sp.) and European polecats (Mustela putorius), using molecular methods. METHODS: In the present study, 174 spleen samples were collected from adult, wild carnivores hunted in the years 2013-2016. A short fragment (383 bp) of the 16S ribosomal RNA gene partial sequence was used as a marker to identify A. phagocytophilum in spleen samples collected from carnivores using nested PCR. RESULTS: The prevalence of A. phagocytophilum in wild carnivores was 31.61% (55/174). Seven sequences of A. phagocytophilum were generated from two raccoon dogs, two badgers, one marten, one red fox and one European polecat. Six identical nucleotide sequences were obtained from one raccoon dog, two badgers, one marten, one red fox and one European polecat (A. phagocytophilum sequences 1: MH328205-MH328209, MH328211), and these were identical to many A. phagocytophilum sequences in the GenBank database (100% similarity). The second sequence (A. phagocytophilum sequence 2: MH328210) obtained from the raccoon dog shared 99.74% identity with A. phagocytophilum sequence 1. CONCLUSIONS: To our knowledge, this is the first study to use molecular methods to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dog, badger, marten and European polecat, in Poland. The detected A. phagocytophilum sequences (1 and 2) were closely related with those of A. phagocytophilum occurring in a wide range of wild and domestic animals and vectors.

Coenurosis of Yak, Bos grunniens, caused by Taenia multiceps: A Case Report with Molecular Identification in Qinghai Tibetan Plateau Area, China.

Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.

High prevalence and genetic diversity of hepatitis B viruses in insectivorous bats from China.

Bats have been identified as the hosts of hepatitis B virus (HBV) in recent years and bats HBV can infect human hepatocyte. We investigated the prevalence and genetic diversity of HBV in bats in China. In this study, a total of 197 insectivorous bats belonging to 10 bat species were captured from karst caves in Mengyin County, Shandong Province and Xianning City, Hubei Province, China. PCR amplification indicated that in total 6.6% (13/197) bats were positive to HBVs. The HBV positive rate in bats was 7.1% (9/127) and 5.7% (4/70) in Shandong Province and Hubei Province, respectively. Phylogenetic analysis indicated that HBV from the two places were in the same cluster with 90.5%-99.5% homology, but distinct from bat HBVs from other places in China and other countries. We concluded that HBV was prevalent and genetic diversified in bats, supporting the hypothesis that bats may be the origin of primate hepadnaviruses.